The present invention is directed to the method of production and therapeutic application of a covalent dimer of the cytokine kit ligand with increased proliferative activity.
Kit ligand (KL) is a growth and differentiation factor for an assortment of cell types, and is known to be a ligand for the c-kit proto-oncogene. KL was initially identified based on a variety of biological activities and has therefore been referred to by different names, including Stem Cell Factor, Mast Cell Growth Factor, and more recently Steel Factor, in recognition of the gene locus in the mouse which encodes KL, as described by Anderson, et al., (1990) Cell 63,235-243; Huang, E., et al. (1990) Cell 63, 225-233; Martin, et al. (1990) Cell 63,203-211; Nocka, et al., (1990) EMBO J. 9, 3287-3294; Williams, et al., (1990) Cell 63,167-174; Zsebo, et al. (1990) Cell 63, 195-201; Zsebo, K. M., et al., (1990) Cell 63, 213-214.
The ability of KL to promote the proliferation of a variety of cell types indicates that KL is useful as a therapeutic in a variety of clinical indications where enhanced hematopoietic recovery would be beneficial. For example, KL stimulates the survival and proliferation of immature hematopoietic stem cells and progenitor cells, as reported by deVries, et al. (1991) J. Exp. Med. 173, 1205; McKniece, et al., (1991) Exp. Hematol. 19, 226-231; Metcalf, et al., Proc. Natl. Acad. Sci. USA 88, 6239-6243; Nocka, et al. (1990) EMBO J. 9, 3287-3294. Thus, KL could be used for the ex vivo expansion of stem cells and progenitors from donor bone marrow prior to transplantation, as proposed in U.S. Pat. No. 5,199,942 to Gillis. KL also acts on erythroid progenitors, and in combination with erythropoietin, drives their differentiation, as reported by Nocka, et al., (1990). This property should make KL useful in treating anemias such as that associated with patients having Diamond Blackfan Syndrome, described by Alter, et al., (1992) Blood 80, 3000-3008. KL is also a potent growth factor for megakaryocytic progenitors and in combination with late acting thrombopoietic factors such as IL-6, stimulates megakaryocytic differentiation, as reported by Briddell (1991), Blood 78, 904-911. KL could thus be useful in stimulating megakaryocyte proliferation and platelet production in thrombocytopenic patients Andrews, et al., (1992) Blood 80, 920-927; Hunt, et al., (1992) Blood 80, 904-911. KL has also been shown to be a potent cytokine in the mobilization of stem cells from the bone marrow to the peripheral blood and, in combination with G-CSF, results in significantly greater numbers of progenitor cells than are mobilized through other treatments, as reported by Andrews, et al., (1992) Blood 80, 920-927; Molineux, et al., (1991) Blood 78, 961; Andrews, et al., (1992) Blood 80, 2715; Briddell, et al., (1993) Blood 82, 1720-1723. Stem cells and progenitors that have first been mobilized and then collected from the peripheral blood have been shown by Juttner, et al. (1992) Int. J. Cell Cloning 10, 160, to be useful either alone or in combination with a bone marrow transplant to speed hematopoietic recovery post radio/chemotherapy.
While KL has many properties which make it a potentially useful therapeutic, KL also acts as a mast cell priming factor and secretagogue, promoting the release of mast cell-derived proinflammatory mediators which can lead not only to local tissue inflammation but more dangerously, to systemic anaphylaxis, as observed by Coleman, et al., (1993) J. Immunol. 150, 556-562; Columbo, et al., (1992) J. Immunol. 149, 599-608; and Nakajima, et al., (1992) Biochem. Biophys. Res. Comm. 183, 1076-1083. The mast cell activating property of KL has been shown to limit the therapeutic potential of native KL. In phase one clinical trials by Amgen of KL administered to patients undergoing chemotherapy, a significant number of patients experienced anaphylactic episodes in response to the KL therapy, mandating their removal from the KL treatment Crawford, et al., (1993) Proc. Am. Soc. Clin. Oncol. 12,135; Demetri, et al., (1993) Proc. Amer. Soc. Clin Oncol. 12, 142. Patients that received lower doses of KL, less than 25 .mu.g/kg/day, exhibited minimal side effects; however, at this dose range, KL alone provides little benefit in terms of hematopoietic recovery or peripheral blood progenitor mobilization.
KL, and the receptor to which it binds, the proto-oncogene c-kit, are considered to be members of the Platelet Derived Growth Factor (PDGF) family. Members of this family have several common features, including the structure of the ligands, described by Nocka, et al., (1990); Flanagan, et al., (1991) Cell 64, 1125-1135; Huang, et al., (1992); Bazan (1991) Cell 65, 9-10; Huang, et al., (1990) and the structure and mechanism of action of the receptors, as described by Williams, et al., (1990) Cell 63, 167-174.
The synthesis and expression of KL is similar to other members of the PDGF family, particularly colony stimulating factor-I (CSF-1 or Macrophage-CSF (M-CSF)) Kawasaki, E. S., et al., (1985) Science 230, 291-296; Wong, G. G., et al., (1987) Science 235, 1504-1508, and the recently identified ligand for the Flt-3/Flk-2 receptor Lyman, et al., (1993) Cell 75, 1157-1167. M-CSF is synthesized from multiple mRNA transcripts that encode for transmembrane proteins, but which lead to either a predominant cell surface bound CSF-1 molecule due to the lack of one proteolytic cleavage site, or to a soluble, proteolytically cleaved CSF-1. Rettenmeier, C. W., Roussel, M. F. (1988) Mol. Cell. Biol. 8, 5026-5034. Similarly, there are at least two naturally occurring forms of KL that arise due to alternative mRNA splicing, as reported by Anderson, et al. (1990), Flanagan, et al., (1991), and Huang, et al., (1992). Both forms are first synthesized as transmembrane proteins. The most abundant form (KL-1) gives rise to a protein of 45 kDa which has a proteolytic cleavage site at amino acids 164-165 (Martin, et al., (1990)), and is readily cleaved to give rise to a soluble protein subunit of 30-35 kDa (Huang, et al., (1992)). The second form of KL (KL-2) is derived from a message in which exon 6, encoding the proteolytic cleavage site, has been spliced out (Anderson, et al., (1990); Flanagan, J. G., et al., (1991) Cell 64, 1125-1135. Without this site a less efficient proteolytic site is used, and the majority of KL-2 remains as a cell surface protein (Flanagan, et al., (1991); Huang, et al, (1992)).
KL does not contain an intermolecular disulfide bond; although it occurs as a dimer when isolated, the units are held together solely by non-covalent interactions (Nocka, et al., (1990); Asakawa, J. Biol. Chem. 266, 18942-18948. Thus, as analyzed by gel filtration chromatography, soluble KL (KL-1) migrates as a dimer of approximately 60 kDa, when glycosylated or 40 kD when not glycosylated. However, when analyzed by SDS-PAGE under reducing or non-reducing conditions, native KL migrates with an apparent molecular weight of a monomer, between 30 and 35 kDa when glycosylated or between 18 and 20 kD when not glycosylated. It is unknown whether membrane associated KL, KL-2, exists in a dimeric state.
cDNA's encoding human, mouse, and rat KL have been cloned and expressed in mammalian, yeast and bacterial cells, as disclosed in PCT/US91/04272 by Immunex Corporation and PCT/US90/05548 by Amgen, Inc. The recombinant KL proteins have biological activity that is comparable to naturally occurring KL of the appropriate species. The protein has been shown to have intrachain disulfide bonds between cysteines at amino acid residues 4 and 89 and at residues 43 and 138, as described by Immunex and Amgen. As described by Amgen, when human KL was expressed as an insoluble protein in E. coli and refolded into active protein, the predominant form of the protein was a properly oxidized protein having a molecular weight of between 18,000 and 20,000 Da as determined under non-reducing conditions. A 37,000 Da protein was also observed under non-reduced conditions; however, no mention of biological activity was made. As reported by Immunex, mutants that were truncated to amino acid 138, that had the first two amino acids removed from the N-terminus, and that were missing the fifth glycosylation site were all active.
Recombinant KL from human and rodent preparations has been found to be as effective as the native molecules when assessed in a variety of in vitro hematopoietic assays. Lu, et al., (1991) J. Biol. Chem. 266, 8102-8107; Martin, et al., (1990) Cell 63,203-211; McKniece, et al., (1991) Exp. Hematol. 19, 226-231. The therapeutic potential of recombinant KL was suggested by its efficacy in several pre-clinical animal models. For example, administration of KL to rodents at dosages of 100 and 200 .mu.g/kg/day led to significant increases in platelets, reticulocytes, and white blood cells, and to a dramatic increase in the number of circulating progenitor cells, as reported by Molineux, et al., (1991) Blood 78, 961; Bodine, et al., (1993) Blood 82, 45-455. Primate studies demonstrated a similar effect of KL on the hematopoietic system, as reported by Andrews, et al., (1991) Blood 78, 1975-1980. An important study in baboons demonstrated a dose-response effect of KL which mirrored effects seen in later clinical trials; KL had little effect on the hematopoietic system at dosages of between 10 and 25 .mu.g/kg/day, but significant effect at between 100 and 200 .mu.g/kg/day, as described by Andrews, et al., (1992) Blood 82, 920-927. Additionally, in a mouse irradiation model, pre-treatment with KL rescued most of the animals exposed to a dose of radiation that was lethal to untreated animals, as described by Zsebo, et al. (1992) Blood 89, 9464-9468.
Although animal models suggested efficacy of KL in stimulating hematopoiesis, when assessed in a clinical trial for its ability to promote the mobilization of stem cells and myeloid progenitors from the bone marrow to the peripheral blood in patients who had received chemotherapy, significant toxicity, manifested as anaphylactic episodes or localized tissue inflammation, occurred in many patients in response to KL, as reported by Crawford, et al., (1993) Proc. Am. Soc. Oncol. 12, 135; Demetri, et al., (1993) Proc. Amer. Soc. Clin. Oncol. 12, 142. This toxicity was attributed to the mast cell priming-degranulating activity of KL, and occurred at dosages of 50 .mu.g/kg/day or greater, below the dosage required for effective stem cell mobilization. Thus, native KL can be considered to possess an unfavorable "P:A" (cell proliferation:mast cell activation) ratio.
It is therefore an object of the present invention to provide a modified form of KL which shows increased potency in mediating cell proliferation in vitro, but no increase in its ability to promote mast cell priming.
It is a further object of the present invention to provide methods for making and using a modified KL having a more favorable P:A ratio which can stimulate hematopoietic recovery or stem cell/progenitor cell mobilization with less toxicity than native KL due to mast cell activation.